Nonlinear microscopy is a powerful technique for investigating cellular processes that underlie normal and disease states in tissue. Existing microscope designs and excitation lasers currently limit the number of imaging channels and the imaging depth. Increasing these will enable studies of more-complex cell interactions in a greater variety of anatomical structures. Recent work has identified the optimal conditions (light pulse energy, duration, wavelength, and repetition rate) for imaging deep in tissue using multiphoton fluorescence and harmonic-generation microscopies. However, sources that supply the needed light pulses and are compatible with bioimaging laboratories are not available. Scientists from Cornell University propose to develop lasers and new microscope designs that will greatly enhance the capabilities for deep-tissue and multi-channel imaging. New concepts in nonlinear pulse propagation will be employed in the design of femtosecond-pulse fiber lasers. These will extend the performance of lasers currently employed in nonlinear microscopy, with the major practical advantages of fiber. Specifically, the sources will supply 100-femtosecond pulses with peak power of 1 megawatt, wavelength-tunable between 800 and 1350 nm and at repetition rates between 1 and 10 MHz. The lasers will ultimately be compact and robust, which will facilitate the use of nonlinear microscopy techniques within and beyond research laboratories. The new lasers will be evaluated through a series of imaging experiments on test samples and through in vivo imaging in the brain and spinal cord of mice. These experiments will also test understanding of the optimum conditions for each imaging situation. A hyperspectral multiphoton microscope that capitalizes on the capabilities of these new lasers will be developed. This will employ multiple excitation and detection wavelengths to vastly increase the amount of information acquired to a total of 80 channels of excitation/emission fluorescence information per pixel. When combined with dense fluorescent labeling strategies, this hyperspectral microscope has the potential to transform in vivo imaging from a tool used primarily for testing hypotheses into a technique for biological discovery.